The present invention is directed to methods for diagnosing and treating cancer patients. In particular, the present invention is directed to methods for determining which patients will most benefit from treatment with inhibitors of receptor protein-tyrosine kinases.
Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body.
A multitude of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. The most commonly used types of anticancer agents include: DNA-alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disrupters (e.g., vincristine, vinblastine, paclitaxel), DNA intercalators (e.g., doxorubicin, daunomycin, cisplatin), and hormone therapy (e.g., tamoxifen, flutamide).
The epidermal growth factor receptor (EGFR) family comprises four closely related receptors (HER1/EGFR, HER2, HER3 and HER4) involved in cellular responses such as differentiation and proliferation. Over-expression of the EGFR kinase, or its ligand TGF-alpha, is frequently associated with many cancers, including breast, lung, colorectal, ovarian, renal cell, bladder, head and neck cancers, glioblastomas, and astrocytomas, and is believed to contribute to the malignant growth of these tumors. A specific deletion-mutation in the EGFR gene (EGFRvIII) has also been found to increase cellular tumorigenicity. Activation of EGFR stimulated signaling pathways promote multiple processes that are potentially cancer-promoting, e.g. proliferation, angiogenesis, cell motility and invasion, decreased apoptosis and induction of drug resistance. Increased HER1/EGFR expression is frequently linked to advanced disease, metastases and poor prognosis. For example, in NSCLC and gastric cancer, increased HER1/EGFR expression has been shown to correlate with a high metastatic rate, poor tumor differentiation and increased tumor proliferation.
Mutations which activate the receptor's intrinsic protein tyrosine kinase activity and/or increase downstream signaling have been observed in NSCLC and glioblastoma. However the role of mutations as a principle mechanism in conferring sensitivity to EGF receptor inhibitors, for example erlotinib (TARCEVA®) or gefitinib (IRESSA™), has been controversial. Recently, a mutant form of the full length EGF receptor has been reported to predict responsiveness to the EGF receptor tyrosine kinase inhibitor gefitinib (Paez, J. G. et al. (2004) Science 304:1497-1500; Lynch, T. J. et al. (2004) N. Engl. J. Med. 350:2129-2139). Cell culture studies have shown that cell lines which express the mutant form of the EGF receptor (i.e. H3255) were more sensitive to growth inhibition by the EGF receptor tyrosine kinase inhibitor gefitinib, and that much higher concentrations of gefitinib was required to inhibit the tumor cell lines expressing wild type EGF receptor. These observations suggests that specific mutant forms of the EGF receptor may reflect a greater sensitivity to EGF receptor inhibitors, but do not identify a completely non-responsive phenotype.
The development for use as anti-tumor agents of compounds that directly inhibit the kinase activity of the EGFR, as well as antibodies that reduce EGFR kinase activity by blocking EGFR activation, are areas of intense research effort (de Bono J. S. and Rowinsky, E. K. (2002) Trends in Mol. Medicine. 8:S19-S26; Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313). Several studies have demonstrated, disclosed, or suggested that some EGFR kinase inhibitors might improve tumor cell or neoplasia killing when used in combination with certain other anti-cancer or chemotherapeutic agents or treatments (e.g. Herbst, R. S. et al. (2001) Expert Opin. Biol. Ther. 1:719-732; Solomon, B. et al (2003) Int. J. Radiat. Oncol. Biol. Phys. 55:713-723; Krishnan, S. et al. (2003) Frontiers in Bioscience 8, e1-13; Grunwald, V. and Hidalgo, M. (2003) J. Nat. Cancer Inst. 95:851-867; Seymour L. (2003) Current Opin. Investig. Drugs 4(6):658-666; Khalil, M. Y. et al. (2003) Expert Rev. Anticancer Ther. 3:367-380; Bulgaru, A. M. et al. (2003) Expert Rev. Anticancer Ther. 3:269-279; Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2:92-313; Ciardiello, F. et al. (2000) Clin. Cancer Res. 6:2053-2063; and Patent Publication No: US 2003/0157104).
Erlotinib (e.g. erlotinib HCl, also known as TARCEVA® or OSI-774) is an orally available inhibitor of EGFR kinase. In vitro, erlotinib has demonstrated substantial inhibitory activity against EGFR kinase in a number of human tumor cell lines, including colorectal and breast cancer (Moyer J. D. et al. (1997) Cancer Res. 57:4838), and preclinical evaluation has demonstrated activity against a number of EGFR-expressing human tumor xenografts (Pollack, V. A. et al (1999) J. Pharmacol. Exp. Ther. 291:739). More recently, erlotinib has demonstrated promising activity in phase I and II trials in a number of indications, including head and neck cancer (Soulieres, D., et al. (2004) J. Clin. Oncol. 22:77), NSCLC (Perez-Soler R, et al. (2001) Proc. Am. Soc. Clin. Oncol. 20:310a, abstract 1235), CRC (Oza, M., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC (Winer, E., et al. (2002) Breast Cancer Res. Treat. 76:5115a, abstract 445). In a phase III trial, erlotinib monotherapy significantly prolonged survival, delayed disease progression and delayed worsening of lung cancer-related symptoms in patients with advanced, treatment-refractory NSCLC (Shepherd, F. et al. (2004) J. Clin. Oncology, 22:14S (July 15 Supplement), Abstract 7022). While most of the clinical trial data for erlotinib relate to its use in NSCLC, preliminary results from phase I/II studies have demonstrated promising activity for erlotinib and capecitabine/erlotinib combination therapy in patients with wide range of human solid tumor types, including CRC (Oza, M., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:196a, abstract 785) and MBC (Jones, R. J., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:45a, abstract 180). In November 2004 the U.S. Food and Drug Administration (FDA) approved TARCEVA® for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) after failure of at least one prior chemotherapy regimen. TARCEVA® is the only drug in the epidermal growth factor receptor (EGFR) class to demonstrate in a Phase III clinical trial an increase in survival in advanced NSCLC patients.
An anti-neoplastic drug would ideally kill cancer cells selectively, with a wide therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, none of the current chemotherapies possess such an ideal profile. Instead, most possess very narrow therapeutic indexes. Furthermore, cancerous cells exposed to slightly sub-lethal concentrations of a chemotherapeutic agent will very often develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents as well. Additionally, for any given cancer type one frequently cannot predict which patient is likely to respond to a particular treatment, even with newer gene-targeted therapies, such as EGFR kinase inhibitors, thus necessitating considerable trial and error, often at considerable risk and discomfort to the patient, in order to find the most effective therapy.
Thus, there is a need for more efficacious treatment for neoplasia and other proliferative disorders, and for more effective means for determining which tumors will respond to which treatment. Strategies for enhancing the therapeutic efficacy of existing drugs have involved changes in the schedule for their administration, and also their use in combination with other anticancer or biochemical modulating agents. Combination therapy is well known as a method that can result in greater efficacy and diminished side effects relative to the use of the therapeutically relevant dose of each agent alone. In some cases, the efficacy of the drug combination is additive (the efficacy of the combination is approximately equal to the sum of the effects of each drug alone), but in other cases the effect is synergistic (the efficacy of the combination is greater than the sum of the effects of each drug given alone).
Target-specific therapeutic approaches, such as erlotinib, are generally associated with reduced toxicity compared with conventional cytotoxic agents, and therefore lend themselves to use in combination regimens. Promising results have been observed in phase I/II studies of erlotinib in combination with bevacizumab (Mininberg, E. D., et al. (2003) Proc. Am. Soc. Clin. Oncol. 22:627a, abstract 2521) and gemcitabine (Dragovich, T., (2003) Proc. Am. Soc. Clin. Oncol. 22:223a, abstract 895). Recent data in NSCLC phase III trials have shown that first-line erlotinib or gefitinib in combination with standard chemotherapy did not improve survival (Gatzemeier, U., (2004) Proc. Am. Soc. Clin. Oncol. 23:617 (Abstract 7010); Herbst, R. S., (2004) Proc. Am. Soc. Clin. Oncol. 23:617 (Abstract 7011); Giaccone, G., et al. (2004) J. Clin. Oncol. 22:777; Herbst, R., et al. (2004) J. Clin. Oncol. 22:785). However, pancreatic cancer phase III trials have shown that first-line erlotinib in combination with gemcitabine did improve survival (OSI Pharmaceuticals/Genentech/Roche Pharmaceuticals Press Release, Sep. 20, 2004).
Several groups have investigated potential biomarkers to predict a patient's response to EGFR inhibitors (see for example, PCT publications: WO 2004/063709, WO 2005/017493, WO 2004/111273, WO 2004/071572, WO 2005/117553 and WO 2005/070020; and US published patent applications: US 2005/0019785, and US 2004/0132097). However, no diagnostic or prognostic tests have yet emerged that can guide practicing physicians in the treatment of their patients with EGFR kinase inhibitors.
The fibroblast growth factor signaling cascade consists of four transmembrane tyrosine kinase receptors (FGFR1-4) and at least 20 different ligands, providing a very complex and context dependent signaling axis. Signaling through this receptor activates a number of important regulators of cell survival and proliferation, including the MAP kinase and PI3K signaling pathways (Omtiz, D M. and Itoh, N. (2001) Genome Biol. 2: 3005). There is a large body of data to indicate that FGFR signaling plays a role in cancer development and progression. FGF receptors are overexpressed in a number of different cancer cell lines derived from a variety of different solid tumors (Chandler, L A., et al., (1999) Int. J. Cancer 81: 451). Functional inhibition of this pathway in these cell lines, by overexpression of dominant negative receptors (Aoki, T. et al., (2002) Int. J. Oncol. 21: 629) or by siRNA or antisense knockdown approaches (Estes Li, N R., et al., (2006) Oncol. Rep. 15: 1407; Yamada, S M., et al., 1999 Glia 28: 66), inhibited the growth and oncogenic potential of the tumor cells. Consistent with these in vitro observations, immunohistochemical staining of tumor tissue sections has indicated high expression of FGF receptors in breast (Wulfing, P., et al., (2005) Br. J. Cancer 92: 1720), prostate (Giri, D F., et al., (1999) Clin. Cancer Res. 5: 1063) and NSCLC (Volm, M R., et al., 1997 Eur. J. Cancer 33: 691) cancer patients. In addition, high FGFR1 expression was shown to be a predictor of poor survival in NSCLC (Volm, M R., et al., 1997 Eur. J. Cancer 33: 691) and pancreatic (Ohta, T., et al., (1995) Br. J. Cancer 72: 824) cancer patients. In further support of these observations Muller-Tudlow and colleagues observed that FGFR1 expression was one of the best predictors of poor survival in NSCLC when compared to a panel of 50 different receptor tyrosine kinases (Muller-Tidow, C., et al. (2005) Cancer Res. 65: 1778). These observations are consistent with the potential role of FGF receptors in more advanced metastatic cancer.
In this context it is interesting to point out that FGFR signaling has been implicated in epithelial to mesenchymal transition (EMT), a process by which cancer cells become more invasive and begin to metastasize. FGF signaling is a key regulator of SNAIL and E-cadherin levels in mouse development (Ciruna, B. and Rossant, J. (2001) Dev. Cell 1: 37) and has also been shown to induce EMT in tumor cells (Valles, A M, et al., (1990) PNAS 87: 1124). FGF-2, in conjunction with TGFβ, was shown to induce an EMT in tubular epithelial cells (Strutz, F., et al., (2002) Endocrinology 146:1145). Of most interest, a combination of FGF-2 and N-cadherin expression increased the metastatic potential of the normally weakly metatstatic breast cancer cell line MCF7 (Hazan, R B., et al., (2000) J. Cell Biol. 148: 779). Taken as a whole these results indicate that FGFR signaling plays a key role in the progression of cancer, potentially through its role in promoting EMT and, as a consequence, metatstasis of tumor cells.
The PDGF receptor signaling network consists of two receptors (PDGFRα and PDGFRβ), which can homo- or hetero-dimerize and 4 ligands (PDGF A-D), which also homo- or hetero-dimerise and differentially activate the different receptor dimers (Pietras, K., et al. (2003) Cancer Cell 3: 439). Ligand occupancy of these receptors results in the activation of a number of different intracellular signaling pathways including Ras and PI3K (Yu, J., et al. (2003) J. Biochem. Mol. Biol. 36: 49). Aberrant signaling by PDGFRs has been associated with cancer development and progression. Amplification of the PDGFRβ gene is associated with high-grade gliomas (Flemming, T P. et al. (1992) Cancer Res. 52: 4550) and gene-fusions involving the PDGFRβ gene have been reported in chronic myelomonocytic leukemia (CMML) (Golub, T R., et al. (1994) Cell 77: 307). Activating point mutations and deletions in the PDGFRα gene have also been reported in GIST patients (Heinrich, M C. et al., (2003) Science 299: 708). In addition increased autocrine PDGFR signaling, through increased expression of PDGF ligand, has been reported to be important in a number of glioma cell lines (Shamah, S M., et al. (1993) Mol. Cell. Biol. 13: 7203). Autocrine PDGFR has also been reported to be important in promoting progression and metastasis of tumors of epithelial origin such as ovarian (Matei, D., et al. (2006) Oncogene 25: 2060) and mammary cancer (Jechlinger, M., et al. (2006) J. Clin. Invest. 116: 1561).
Although these lines evidence support a role for the PDGF receptors in primary tumor biology, a key role for these receptors in cancer is in the regulation of the tumor microenviroment through paracrine signaling. PDGFRs are classically restricted to cells of a mesenchymal lineage and so are expressed in connective tissue fibroblasts. The expression of PDGF ligand from tumor cells acts as both a chemo attractant and a mitogen for PDGFR expressing stromal cells, leading to primary tumor development and promotion of angiogenesis. For example the growth of a NSCLC cell line Calu6, in a xenograft setting, was dependent upon tumor cell production of PDGF-AA allowing recruitment of mouse stromal cells to support primary tumor growth (Tejada, M L., et al. (2006) Human Cancer Biol. 12: 2676). Further, the potential importance of paracrine PDGFR signaling in advanced breast cancer was suggested by the observation that PDGF-BB ligand expression was restricted to tumor epithelial cells, whereas it was predominately the stromal cells adjacent to the tumor that expressed high levels of PDGF receptor (Coltrera et al., 1995). Therefore the paracrine signaling mediated by PDGFRs may be key in regulating the microenvironment to allow conditions supportive for primary tumor growth and the establishment of metastases at distant organ sites.
Another interesting, and related, aspect of PDGFR biology is that it has been implicated in EMT. Treatment of an epithelial colorectal cancer cell line, HT29, with PDGF induced all the hallmarks of a full EMT (Yang, L., et al. (2006) Cell 127: 139). In addition mouse epithelial cells stimulated with TGFβ show a marked increase in expression of PDGFRα, PDGFRβ and PDGF-AA. Signaling through this axis was crucial in maintaining the mesenchymal-like phenotype (Jechlinger, M., et al. (2006) J. Clin. Invest. 116: 1561). Taken together, these data indicate that PDGFR signaling either in an autocrine or paracrine fashion plays an important role in the development and progression of many different cancer types. These include the promotion of stromal cell recruitment and angiogenesis to provide a suitable tumor microenviroment and the stimulation and maintenance of EMT to promote tumor progression and metastasis.
During most cancer metastases, an important change occurs in a tumor cell known as the epithelial-mesenchymal transition (EMT) (Thiery, J. P. (2002) Nat. Rev. Cancer 2:442-454; Savagner, P. (2001) Bioessays 23:912-923; Kang Y. and Massague, J. (2004) Cell 118:277-279; Julien-Grille, S., et al. Cancer Research 63:2172-2178; Bates, R. C. et al. (2003) Current Biology 13:1721-1727; Lu Z., et al. (2003) Cancer Cell. 4(6):499-515)). Epithelial cells, which are bound together tightly and exhibit polarity, give rise to mesenchymal cells, which are held together more loosely, exhibit a loss of polarity, and have the ability to travel. These mesenchymal cells can spread into tissues surrounding the original tumor, as well as separate from the tumor, invade blood and lymph vessels, and travel to new locations where they divide and form additional tumors. EMT does not occur in healthy cells except during embryogenesis. Under normal circumstances TGF-β acts as a growth inhibitor. However it is believed that during cancer metastasis, TGF-β begins to promote EMT.
Thus, there remains a critical need for improved methods for determining the best mode of treatment for any given cancer patient and for the incorporation of such determinations into more effective treatment regimens for cancer patients, whether such inhibitors are used as single agents or combined with other anti-cancer agents.